Multi-dose erythropoietin formulations

ABSTRACT

The present invention is directed to multi-dose erythropoietin formulations for parenteral administrations. The formulations contain one or more of the following preservatives benzethonium chloride, phenoxyethanol and phenylethyl alcohol.

BACKGROUND OF THE INVENTION

I. Field of the Invention

The present invention pertains to multi-dose formulations oferythropoietin (hereinafter “EPO”), comprising a particularlyadvantageous preservative or combination of preservatives. Specifically,the present invention pertains to the use of the preservativesbenzethonium chloride, phenoxyethanol and phenylethyl alcohol, alone orin combination, in multi-dose EPO formulations. The invention disclosedherein also relates to a method of treating a patient with anEPO-containing composition comprising benzethonium chloride,phenoxyethanol and/or phenylethyl alcohol. The present invention furtherrelates to a pharmaceutical carrier composition; a vial for containing acomposition; and a method of inhibiting microbial growth in a solution;wherein all compositions or solutions comprise EPO and one or more ofthe preservatives benzethonium chloride, phenoxyethanol and phenylethylalcohol.

II. Background of the Invention

Sterility is one of the most important characteristics of parenteralproducts. For parenteral products that are sterilized and intended forsingle dose injection, maintenance of sterility is a function of boththe method of sterilization and the integrity of the packaging system.For parenteral products that are intended for multiple dosing,antimicrobial agents must be added to the product formulation to protectthe product from accidental microbial contamination during its storageand/or use.

Stable protein-containing multi-dose pharmaceutical formulations areviewed by the pharmaceutical industry as particularly advantageous andcommercially attractive. Multi-dose formulations are generally, thoughnot always, contained in vials (multi-dose containers) that allow forthe extraction of partial amounts of the formulation at various times.This type of system is desirable as it allows multiple doses to beobtained from a single container, and allows for more controlledadministration of the pharmaceutical composition as the formulation maybe withdrawn and administered in any partial amount.

The nature of the use of multi-dose formulations imposes specialrequirements on the formulation. For example, maintenance of thesterility of the composition is particularly challenging given the manyopportunities for introduction of microorganisms and other contaminantsinto the formulations. Repeated introduction of foreign elements, forexample, needles, into the multi-dose container after formulationcreates a likelihood of introducing microorganisms into the container.Additionally and alternatively, microorganisms may be introduced duringfilling of the containers, or during reconstitution of the formulationsafter lyophilization and prior to administration. The extended periodsof time over which the container may be stored—especially duringmultiple introductions of foreign elements, and/or after contaminantsmay have been introduced, demands that the formulation contain specialadditives to insure the sterility of the contents.

To insure that multi-dose formulations maintain optimally sterileproperties, the United States Food and Drug Administration (FDA) andregulatory agencies in other jurisdictions require that all multi-doseformulations contain preservatives to prevent the growth of, or toaffirmatively kill, any microorganisms that may be introduced into theformulations. Given the inherent instability of proteins, and theirtendency to interact adversely with preservative compounds, thedevelopment of protein containing multi-dose formulations has provenparticularly difficult. Possible adverse interactions betweenpreservatives and proteins include the degradation of the protein,especially when stored for extended periods of time; inactivation of theprotein; formation of protein aggregates; and other interactions thatinactivate the formulation or make administration of the formulation tohumans, particularly by infusion, injection or other parenteraladministration, difficult, painful or otherwise undesirable.

Additionally, preservatives themselves are noted for causing acuteadverse reactions, such as allergic reactions, in humans upon parenteraladministration. Ideally, the preservative contained in the multi-doseprotein pharmaceutical composition should be effective in lowconcentration against a wide variety of micro organisms, soluble in theformulation, non-toxic, compatible and non-reactive with the protein,active with long term stability, and non-reactive with components of thecontainer or closure system.

Sandeep Nema et al. published lists of various excipients that have beenincluded in the formulation of injectable products marketed in the USA.The antimicrobial preservatives listed in this review article areincluded in Table 1: TABLE 1 ANTIMICROBIAL PRESERVATIVES PreservativeFrequency Range Benzalkonium chloride 1 0.02% w/v Benzethonium chloride4 0.01% Benzyl alcohol 74 0.75-5% Chlorobutanol 17 0.25-0.5% m-cresol 30.1-0.3% Myristyl gamma-picolinium 2 0.0195-0.169% chloride Parabenmethyl 50 0.05-0.18% Paraben propyl 40 0.01-0.1% Phenol 48 0.2-0.5%2-Phenoxyethanol 3 0.50% Phenyl mercuric nitrate 3 0.001% Thimerosal 460.003-0.01%

EPO is a glycoprotein that functions to stimulate the production ofhemoglobin and erythrocytes in bone marrow. It is produced in thekidney, and is widely used as a treatment for anemia caused by a varietyof conditions, including, for example, renal failure. The amino acidsequence and general glycosylation patterns of EPO are known in the art.See, for example, Miyaka et al. and U.S. Pat. No. 4,703,008. Isolationand Purification of EPO, from human tissues or fluids, has beendescribed by Miyake et al.

The nucleic acid sequence encoding the protein, isolation of thissequence, and production of the protein by traditional recombinationmethods are also known in the art. See, for example, U.S. Pat. No.4,703,008 to Lin, describing the nucleic acid sequence encoding EPO;U.S. Pat. No. 4,337,513 to Sugimoto et al., describing the use oflymphoblastoid cells to produce EPO; and Sherwood et al., describingproduction of EPO by a human renal carcinoma cell line. Additionally,production, isolation and purification of the protein is also achievableby gene-activation, or homologous recombination, followed by well-knownisolation and purification techniques.

Development of EPO-containing multi-dose formulations has provenparticularly difficult by virtue of the particular instability displayedby EPO, and its tendency to readily interact with common pharmaceuticalingredients. U.S. Pat. No. 4,806,524. Attempts to develop multi-dose EPOformulations have tried to circumvent these problems by maintaining theformulations at a low pH, or by including various amino acid constructs,two approaches thought to assist in the stabilization of the EPOprotein, or by developing lyophilized forms in which the preservativesublimes from the formulation before administration. U.S. Pat. No.5,503,827 (the '827 patent) to Woog.

Stable, sterile multi-dose EPO-containing pharmaceutical formulationsare few. They include those formulations disclosed in the '827 patent.The '827 reference discloses and specifically claims chloretone(chlorbutanol, 1,1,1-trichloro-2-methyl-2-propanol), benzalkoniumchloride or benzyl alcohol as preservatives. Woog specifically notes theparticular difficulty of providing a multi-dose EPO formulation in whichthe allergy rates are reduced, and promotes the use of the specificallyclaimed preservatives as especially advantageous in that regard. Thisreference further stresses that due to the tendency of preservatives todegrade and proteins to be inactivated when combined, it is mostdesirable to minimize contact between the preservative and the protein.The '827 patent further discloses the use of several amino acidconstructs and other additives thought necessary to stabilize EPO insolution. Finally, The '827 patent discloses, in a most preferredembodiment that any preservative used in the initial formulation issublimed away upon lyophilization of the composition. Then, uponreconstitution, additional preservative selected from the groupdisclosed (chloretone) as defined, benzalkonium chloride and benzylalcohol, may be introduced, but the injectable, reconstituted solutionshould be used within 30 days.

Another example of an EPO-containing multi-dose formulation is describedin U.S. Pat. No. 5,661,125 (the '125 patent). This patent explicitlyacknowledges and affirms other references stating that EPO “is aninstable substance especially in solution form” and “when combined withknown stabilizers, the resulting stability of the EPO is varied andunpredictable.” This reference then goes on to show and claim thespecific use of benzyl alcohol, a paraben and/or a phenol or acombination of these as a preservative in EPO-containing solutions.Further attesting to the difficulty of discerning compatible andadvantageous preservatives for use in EPO-containing multi-doseformulations, this reference states:

-   -   “ . . . nothing specific can be derived from the use of        preservatives with other proteins that would suggest any        particular preserved formulation for erythropoietin. See, e.g.,        Geigert, J., ‘Overview of the Stability and Handling of        Recombinant Protein Drugs,’ Journal of Parenteral Science &        Technology, Vol. 43(5):220-224 (1989)”.

Accordingly, there remains a need for an EPO-containing, preserved,multi-dose pharmaceutical formulation that: (1) maintains the stabilityof the protein component and the composition over an extended shelf lifeof the product; (2) maintains the sterility of the formulation and meetsthe United States, European and Japanese Pharmacopia criteria forpreservative challenge testing; (3) is safe in the concentrations used;and (4) is administrable—by any parenteral or oral route—in a mannerthat is effective, and minimizes pain and the chance of adversereaction, for example, allergic reaction, in the patient.

III. SUMMARY OF THE INVENTION

The present invention provides a novel and particularly advantageousmulti-dose formulation containing erythropoietin and the preservativesbenzethonium chloride, phenoxyethanol and phenylethyl alcohol, eitheralone or in combination.

The formulations of the present invention may be formulated in a varietyof concentrations in various vial sizes for various administrationdosages. For example, the formulations disclosed in the presentinvention may comprise 10,000, 20,000, 40,000, or even up to or greaterthan 100,000 Unit/ml EPO concentrations. They may further contain anyconcentrations in between these exemplary concentrations, such as 5,000,15,000, 25,000 unit/ml concentrations, and the like. Additionally, thedosages may be formulated in a ½, 1 or 2 ml vial, or any other size vialor other container preferred by the formulator. It will be clear to oneof skill in the art, that any combination of dosages and vials may beused, depending upon the needs of the formulator. For example, one couldprepare the presently disclosed formulations as a 10,000 unit/½ mlconcentration in a 1 ml vial, a 40,000 unit/ml dose in a 2 ml vial, orany other combination of concentration of EPO in any size vial. Thecompositions may be in the form of an aqueous solution, a suspension, ormay be lyophilized.

The present invention provides in an alternative embodiment, apharmaceutical carrier composition, for use as a carrier of EPO,comprising the preservatives benzethonium chloride, phenoxyethanol, orphenylethyl alcohol, wherein the preservatives are contained in the EPOcarrier composition alone or in combination. The present invention alsoprovides a vial for containing multiple doses of EPO, wherein the vialcomprises EPO and an effective amount of one or a combination of thefollowing preservatives: benzethonium chloride, phenoxyethanol, andphenylethyl alcohol.

In yet another embodiment, the present invention provides a method ifinhibiting microbial growth in an EPO-containing solution, wherein themethod comprises adding to the EPO-containing solution, one or acombination of the following preservatives: benzethonium chloride,phenoxyethanol, and phenylethyl alcohol.

Additional components of the EPO multi-dose formulations of the presentinvention include surfactants, buffers, osmolality adjusting agents andantiadsorbants. Particularly advantageous additives includepolysorbate-20, polysorbate-80, sodium phosphate, sodium chloride andgenapol.

The formulations of the present invention may be in solid, semi-solid,liquid or fluid form, for example, as tablet, aqueous solution or asuspension, or may be lyophilized and reconstituted prior toadministration to a patient. The formulations may be administered viaany parenteral route, including intravenous, subcutaneous,intramuscular, transdermal, intra-arterial, intra-peritoneal, or viapulmonary inhalation. They may also be administered orally.

IV. DETAILED DESCRIPTION OF THE INVENTION

The present invention provides a significant improvement over the stateof the art. Provided are novel EPO-containing multi-dose pharmaceuticalformulations containing preservatives that individually provide forstable, sterile, easily administered compositions. Further, and mostunexpectedly, the present invention discloses that phenoxyethanol andbenzethonium chloride, when used in combination in an EPO-containingmulti-dose pharmaceutical composition, have positive synergistic effectsresulting in a particularly advantageous composition. Specifically, thiscombination of preservatives displays the following characteristics: (1)synergistic antimicrobial effect, allowing for a lower concentration ofpreservatives to be used; (2) excellent stability of the EPO, at varyingstorage conditions, over extended periods of time; and (3)phenoxyethanol has a potential for a local anesthetic effect, making thecomposition particularly preferable for subcutaneous administration.

As used herein, the following terms have the following meanings:

Erythropoietin—a glycoprotein which, when in biologically active andglycosylated form, has the capacity to induce the formation ofhemoglobin and red blood cells in bone marrow. May be obtained viaisolation from human tissues or fluids, by traditional recombinationmethods, or by gene activation.

Parenteral—by some means other than the gastrointestinal tract; includesintravenous, subcutaneous, intramuscular, and intramedullary,intra-arterial, intra-peritoneal and pulmonary inhalation.

Pharmaceutically acceptable (or pharmacologically acceptable)—refers tomolecular entities and compositions that do not produce an adverse,allergic or other untoward reaction when administered to an animal or ahuman, as appropriate.

Pharmaceutically acceptable carrier—includes any and all solvents,dispersion media, coatings, antibacterial and antifungal agents,isotonic and absorption delaying agents and the like, that may be usedas a media for a pharmaceutically acceptable substance.

Unit—a unit of biological activity as determined by exhypoxicpolyeythemic mouse bioassay and compared to World Health Organizationstandards.

Any numerical values recited herein include all values from the lowervalue to the upper value in increments of one unit provided that thereis a separation of at least 2 units between any lower value and anyhigher value. As an example, if it is stated that the concentration of acomponent or a value of a process variable such as, for example,osmolality, temperature, pressure, time and the like is, for example,from 1 to 90, preferably from 20 to 80, more preferably from 30 to 70,it is intended that values such as 15 to 85, 22 to 68, 43 to 51, 30 to32 etc. are expressly enumerated in this specification. For values whichare less than one, one unit is considered to be 0.0001, 0.001, 0.01 or0.1 as appropriate. These are only examples of what is specificallyintended and all possible combinations of numerical values between thelowest value and the highest value enumerated are to be considered to beexpressly stated in this application in a similar manner.

A. Preservatives: Phenoxyethanol and Benzethonium Chloride

The preservatives contemplated for use according to the presentinvention are preferably benzethonium chloride, phenoxyethanol andphenylethyl alcohol, any variants of these preservatives and theirstructural analogues. It is specifically contemplated that any of thesepreservatives may be used as the sole preservative in the presentlydisclosed formulations, or they may advantageously be used incombination with each other. As shown herein, formulations of thepresent invention using a combination of phenoxyethanol and benzethoniumchloride prove particularly preferable.

Benzethonium chloride, phenoxyethanol and phenylethyl alcohol may beused in the presently disclosed formulations in any effective amount.The total preservative concentration is preferably between about 0.001%and about 4.0% of the total formulation. Particularly advantageousconcentrations of total preservative are those maintained as low aspossible to achieve the requisite antimocrobial effect, while minimizingthe potential for adverse reactions.

When benzethonium chloride is used as the sole preservative, it isadvantageously employed in concentrations of from about 0.001 to about1.0%. More preferably, benzethonium chloride, when used as the solepreservative, may be employed in concentrations ranging from about 0.01to about 0.5%, or 0.02 to about 0.8%, and most preferably in aconcentration of from about 0.01 to 0.02% of the total composition.

When phenoxyethanol is used in the absence of other preservatives suchas benzethonium chloride, the EPO formulation will meet United Statesregulatory requirements when the phenoxyethanol content is 0.5%.Preferable concentrations of phenoxyethanol range from about 0.1 toabout 3.0%, and more preferably from about 0.25 to about 2.0%. Mostpreferable concentrations of phenoxyethanol, when used as the solepreservative, range from about 0.5% to about 1.0%. When phenylethylalcohol is used as the sole preservative, preferred concentrations arefrom about 0.01 to about 2.0%. Preferred concentrations range from about0.1 to about 1.0%, with most preferred concentrations ranging from about0.25 to about 0.5%.

In more preferred embodiments of the present invention, bothbenzethonium chloride and phenoxyethanol are used together.Surprisingly, when used together these preservatives have a synergysticeffect on one another, and may both be used in concentrations lower thanwould be required if used alone. Preferred formulations includebenzethonium chloride in concentrations of from about 0.001 to about0.1% in combination with phenoxyethanol in concentrations of from about0.01 to about 1.0%. More preferred formulations contain benzethoniumchloride in a concentration of from about 0.01% to about 0.02% andphenoxyethanol in a concentration of from about 0.25% to about 0.5%.

In another embodiment, the present invention includes benzethoniumchloride in combination with phenylethyl alcohol. Preferred formulationsinclude benzethonium chloride in concentrations of from about 0.001 toabout 0.1% together with phenylethyl alcohol in concentrations of fromabout 0.01 to about 1.0%. More preferred formulations containbenzethonium chloride in a concentration of from about 0.15 to about0.25% and phenylethyl alcohol in a concentration of from about 0.2 toabout 0.5%. A most preferred formulation in which benzethonium chlorideand phenylethyl alcohol are used in concert, includes benzethoniumchloride in a concentration of about 0.02% and phenylethyl alcohol in aconcentration of about 0.25%.

B. Erythropoietin

The nucleic acid sequence, amino acid sequence, three-dimensionalstructure, and typical glycosylation patterns of EPO are known in theart. Isolated and purified EPO from various sources is also known.Accordingly, one of skill in the art can obtain EPO for use according tothe present invention by isolating and purifying the EPO from humantissue or fluids, through traditional recombinant techniques, andthrough gene activation processes. All of these methods are specificallycontemplated to be within the scope of this patent. Additionally, anyother EPO, obtained from any source, is contemplated for use accordingto the present invention.

C. Other Active Components

The optimal formulation according to the present invention may varyaccording to factors such as amount of time the formulation will bestored, conditions under which it will be stored and used, theparticular patient population to which it may be administered, etc.Adjustments to the formulation by adjusting constituents of theformulations and their relative concentrations, other than thepreservatives benzethonium chloride, phenoxyethanol and phenylethylalcohol and EPO as described supra, may be made as needed according tothe needs of the formulator, administrator or patient. Additionalconstituent elements of the multi-dose EPO formulations of the presentinvention may include water, a buffer, a surfactant or antiadsorbant, awetting agent, and an osmolality adjusting agent. Formulationcharacteristics that may be modified include, for example, the pH andthe osmolality, to achieve a formulation that has a pH and osmolalitysimilar to that of human blood or tissues.

Buffers are useful in the present invention for, among other purposes,manipulation of the total pH of the pharmaceutical formulation. Avariety of buffers known in the art may be used in the presentformulations, such as various salts of organic or inorganic acids,bases, or amino acids, and including various forms of citrate,phosphate, tartrate, succinate, adipate, maleate, lactate, acetate,bicarbonate, or carbonate ions. Particularly advantageous buffers foruse in the present invention include sodium or potassium buffers,particularly sodium phosphate. In a preferred embodiment, sodiumphosphate is employed in a concentration approximating 20 mM. Aparticularly effective sodium phosphate buffering system comprisessodium phosphate monobasic monohydrate and sodium phosphate dibasicheptahydrate. When this combination of monobasic and dibasic sodiumphosphate is used, advantageous concentrations of each are about 0.5 toabout 1.5 mg/ml monobasic and about 2.0 to about 4.0 mg/ml dibasic, withpreferred concentrations of about 0.9 mg/ml monobasic and about 3.4mg/ml dibasic phosphate. The pH of the formulation changes according tothe amount of buffer used. It is preferred to achieve a pH level ofbetween 5.0 and 8.0, more preferable to have a pH of about 6.0 to about7.5, and most preferable to develop a formulation with a pH of about7.0.

It may also be advantageous to employ surfactants in the presentlydisclosed formulations. Surfactants or anti-adsorbants that prove usefulaccording to the present invention include polyoxyethylenesorbitans,polyoxyethylenesorbitan monolaurate, polysorbate-20, such as Tween-20™,polysorbate-80, hydroxycellulose, and genapol. In a preferredembodiment, polysorbate-20 is used. When any surfactant is employed inthe present invention, it is advantageous to use it in a concentrationof about 0.01 to abut 0.5 mg/ml. In a particularly useful embodiment,polysorbate-20 is used in a concentration of about 0.1 mg/ml.

Additional useful additives are readily determined by those of skill inthe art, according to particular needs or intended uses of the disclosedmulti-dose EPO formulations. One such particularly useful additionalsubstance is sodium chloride, which is useful for adjusting theosmolality of the formulations to achieve the desired resultingosmolality. Particularly preferred osmolalities are in the range ofabout 270 to about 330 mOsm/kg. The optimal osmolality of the presentlydisclosed formulations is approximately 300 mOsm/kg. Sodium chloride inconcentrations of about 6.5 to about 7.5 mg/ml are affective forachieving this osmolality, with a sodium chloride concentration of about7.0 mg/ml being particularly effective. Or the amount of sodium chloridecan be added or adjusted to achieve an osmolality of about 270 to about330 mOsm/kg, and preferably 300 mOsm/kg. Other useful osmolalityadjusting agents include mannitol and sorbitol.

D. Preparation of the Compositions

The EPO formulations described herein may be prepared in water suitablymixed with a surfactant, such as hydroxypropylcellulose orpolyoxyethylenesorbitans. In many cases, it will be preferable toinclude isotonic agents, for example, sugars or sodium chloride asdescribed above. Prolonged absorption of the injectable compositions canbe brought about by the use in the compositions of agents delayingabsorption, for example, aluminum monostearate or gelatin. Other agentsthat may be employed include, but are not limited to lecithin, urea,ethylene oxide, propylene oxide, hydroxypropylcellulose,methylcellulose, or polyethylene glycol.

Aqueous compositions (inocula) as described herein may include aneffective amount of EPO dissolved or dispersed in a pharmaceuticallyacceptable aqueous medium. Such compositions are also referred to asinocula. The use of pharmaceutically acceptable carrier media and agentsfor pharmaceutically active substances is well known in the art. Exceptinsofar as any conventional media or agent is incompatible with theactive ingredient, its use in the therapeutic compositions iscontemplated. Supplementary active ingredients also can be incorporatedinto the compositions as described above.

A proteoglycan such as EPO may be formulated into a composition in aneutral or salt form. Pharmaceutically acceptable salts include the acidaddition salts (formed with the free amino groups of the protein) andthose that are formed with inorganic acids such as, for example,hydrochloric or phosphoric acids, or such organic acids as acetic,oxalic, tartaric, mandelic, and the like. Salts formed with the freecarboxyl groups can also be derived from inorganic bases such as, forexample, sodium, potassium, ammonium, calcium, or ferric hydroxides, andsuch organic bases as isopropylamine, trimethylamine, histidine,procaine and the like.

The therapeutic compositions of the present invention are advantageouslyadministered in the form of injectable compositions either as liquidsolutions or suspensions; solid forms suitable for solution in, orsuspension in, liquid prior to injection may also be prepared. A typicalcomposition for such purposes comprises a pharmaceutically acceptablecarrier. For instance, the composition may contain 10 mg, 25 mg, 50 mgor up to about 100 mg of human serum albumin per milliliter of phosphatebuffered saline

The formulations as described herein may be contained in a vial, bottle,tube, syringe or other container for single or multiple administrations.Such containers may be made of glass or a polymer material such aspolypropylene, polyethylene, or polyvinylchloride, for example.Preferred containers may include a seal, or other closure system, suchas a rubber stopper that may be penetrated by a needle in order towithdraw a single dose and then re-seal upon removal of the needle. Allsuch containers for injectable liquids, lyophilized formulations,reconstituted lyophilized formulations or reconstitutable powders forinjection known in the art are contemplated for use in the presentdisclosed compositions and methods.

V. EXAMPLES

The following examples are illustrative only and are not to be construedas intended to limit the scope of the invention.

A. Example 1 Preservative Selection and Stability Testing

Materials and Methods:

Sodium phosphate monobasic monohydrate USP, sodium phosphate dibasicheptahydrate USP, sodium chloride USP/EP, and polysorbate 20 USP/NF wereobtained from J.T. Baker, a division of Mallinckrodt Baker, Inc.,Phillipsburg, N.H. 08865.

Benzalkonium chloride USP/NF, 2-phenoxyethanol BP, phenylethyl alcoholUSP/NF, thimerosal USP/NF, phenol crystals USP, benzethonium chlorideUSP/NF, m-cresol USP, phenyl mercuric nitrate USP/NF, benzyl alcoholUSP, chlorobutanol USP, methylparaben USP/NF, and propylparaben USP/NFwere obtained from Spectrum Quality products INC., Gardena, Calif.90248.

Myristyl gamma-picolinium chloride was obtained from Pharmacia & UpjohnCompany, Kalamazoo, Mich. 49001.

Multi-dose EPO-containing solutions were formulated as sterile,non-pyrogenic, colorless aqueous solutions in water for injection at10,000 units and 20,000 units concentration. Solutions preparedcontained a 20 mM phosphate buffer (sodium phosphate monobasicmonohydrate, and sodium phosphate dibasic heptahydrate), 0.01% W/Vpolysorbate 20 as an antiadsorbent, 0.45-0.8% w/v sodium chloride(depending on the preservative system that was used, the amount ofsodium chloride was adjusted to produce an Osmolality of approximately300 mOsm/kg), and a preservative system. Solutions prepared had a pH ofapproximately 7.0, and an Osmolality of approximately 300 mOsm/kg.

Solutions were sterilized by filtration through a sterile 0.22-micronMillipore filter. Solutions were packaged in sterile, clear 2 ml USPtype 1 glass vials and stored at 5° C. and 25° C. for chemical stabilitytesting and in sterile 250-500 ml HDPP (high density polypropylene)bottles for microbial testing.

Preservatives studied included: benzyl alcohol 1.0% w/v, benzalkoniumchloride 0.01% w/v, 2-phenoxyethanol 0.5% w/v, phenylethyl alcohol 0.5%w/v, thimerosal 0.005% and 0.01% w/v, phenol crystals 0.4% w/v,benzethonium chloride 0.01% and 0.02% w/v, m-cresol 0.4% w/v, phenylmercuric nitrate 0.002% w/v, methylparaben 0.1 and 0.18% w/v, andpropylparaben 0.03% and 0.035% w/v, and myristyl-gamma-picoliniumchloride 0.02% w/v. Also, the following combinations were studied: (1)benzethonium chloride 0.005% w/v with phenoxyethanol 0.25% w/v; (2)benzethonium chloride 0.005% and phenoxyethanol 0.5% w/v; (3)benzethonium chloride 0.01% w/v with phenoxyethanol 0.5% w/v; and (4)phenylethyl alcohol 0.25% w/v and benzethonium chloride 0.02% w/v.

Discussion:

Methyl paraben, propyl paraben, m-cresol, and phenol produced hazy tocloudy solutions when added to the EPO formulation (buffered solution).This cloudiness problem was identified as an incompatibility between theabsorbent polysorbate 20, and each of these preservatives (Handbook ofPharmaceutical Excipients, 1994).

Although chlorobutanol produced a clear solution when used in theformulation, its evaluation was stopped because it is not stable atpH >3, its half-life at pH 7.5 is approximately 3 months (Handbook ofPharmaceutical Excipients, 1994).

In the presence of phenyl mercuric nitrate, a cloudy solution wasproduced. This cloudiness was identified as an incompatibility betweenthe osmotic agent sodium chloride, and phenyl mercuric nitrate (Handbookof Pharmaceutical Excipients, 1994).

Thimerosal produced a clear solution when used in the EPO formulation.Also, it showed good preservative efficacy. Additionally, the EPOformulation showed good chemical stability in the presence ofthimerosal. However, since it contains mercury, its use is likely to beunacceptable by the agencies in Europe, Japan and US.

Formulations containing benzyl alcohol, benzethonium chloride,phenoxyethanol, phenylethyl alcohol, benzalkonium chloride, andmyristyl-gamma-picolinium chloride produced clear solutions. Based onthe minimum inhibitory concentrations for these preservatives, safety,and frequency of their use, the following preservatives were selectedfor further chemical evaluation of the protein stability andanti-microbial effectiveness of the EPO formulation: benzyl alcohol,benzethonium chloride, phenoxyethanol, phenylethyl alcohol, and severalbenzethonium chloride & phenoxyethanol combinations.

B. Example 2 Stability of Protein Tested

Materials and Methods:

Prototype batches were made using these selected preservatives andplaced on stability at 5° C. and 25° C. Samples were tested by a reversephase HPLC method. The assay results (% label claim) of the EPOcontaining formulations in the presence of these preservatives are shownin Table 2.

Label claim was determined by reverse phase HPLC using a WatersDelta-Pak™ C18 column and gradient elution using an aqueous solutioncontaining 0.05% TFA and acetonitrile concentration which increases from23 to 86%. Detection of the EPO protein was monitored at 210 nm. TABLE 2ASSAY RESULTS IN TERMS OF CONCENTRATION (% LABEL CLAIM) Chemical Assay(% LC)¹ Preservative Months Strength 5° C. 25° C. Benzyl Alcohol 7 M10,000 U/ml 98.6 90.0 Benzyl Alcohol 7 M 20,000 U/ml 99.7 93.7Benzethonium 4 M 10,000 U/ml 99.9 88.9 Benzethonium 4 M 20,000 U/ml101.5 94.4 Phenoxyethanol 4 M 10,000 U/ml 100.5 96.9 Phenoxyethanol 4 M20,000 U/ml 102.6 97.5 Benzethonium & 3 M 20,000 U/ml 98.3 97.0Phenoxyethanol (0.005% & 0.5%)¹as measured by reverse phase HPLC.

As can be seen from table 2, the reverse-phase HPLC data show no loss ofconcentration of EPO for all formulas when stored at 5° C. for up to 3-7months. However, formulas containing benzyl alcohol or benzethoniumchloride alone showed up to 10% loss of EPO when stored at 25° C. for upto 3-7 months. Formulas containing phenoxyethanol or phenoxyethanol &benzethonium chloride in combination showed no loss of EPO when storedat 25° C. for up to 3-4 months. These results show the stabilizingeffect of phenoxyethanol and phenoxyethanol & benzethonium chloride incombination on EPO, this effect is surprising and unexpected as well asextremely advantageous.

C. Example 3 Preservative Challenge Test

Preservative effectiveness tests are Compendial-guided assays thatdetermine efficacy for preservative systems in multi-dose pharmaceuticalpreparations. In such assays, test formulations are challenged withstandardized suspensions of indicator aerobic bacteria and molds andmicroorganism survival is monitored over a 28-day period.

Table 3 shows the results of the United States Pharmacopia (USP) andEuropean Pharmacopia (EP) preservative challenge testing. All testedformulas passed the USP criteria for preservative challenge test.Formulas containing 0.01% w/v benzethonium chloride, 0.5% w/vphenoxyethanol, or 0.5% w/v phenylethyl alcohol failed the EP criteriafor preservative challenge test. Formulas containing benzethoniumchloride and phenoxyethanol in different combinations and benzethoniumchloride and phenylethyl alcohol in combination passed both the USP andthe EP criteria. Based on the data shown in table 3, it appears that theantimicrobial activity of benzethonium chloride was surprisinglyincreased by the addition of phenoxyethanol, in a synergistic manner.TABLE 3 PRESERVATIVE CHALLENGE TESTING Preservative ChallengePreservative Strength USP EP Benzyl Alcohol 20,000 U/ml passed not done(1.0%) Benzethonium 20,000 U/ml (0.01%) passed failed (0.02%) passedpassed Phenoxyethanol 20,000 U/ml passed failed (0.5%) Benzethonium &20,000 U/ml passed passed Phenoxyethanol (0.005 & 0.25%) (0.005 & 0.5%)(0.01 & 0.5%) Phenylethyl Alco. 20,000 U/ml passed failed 0.5%Benzethonium & 20,000 U/ml passed passed Phenylethyl Alco (0.02% &0.25%)

REFERENCES

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1. A pharmaceutical composition comprising a stable, sterile, multi-doseformulation of erythropoietin and an amount of benzethonium chlorideeffective to inhibit microbial growth in said composition.
 2. Thecomposition of claim 1, wherein the composition further comprisesphenoxyethanol.
 3. The composition of claim 1, wherein the compositionfurther comprises phenylethyl alcohol.
 4. The composition of claim 1,wherein an effective amount of benzethonium chloride is a concentrationof from about 0.001 to about 1.0% w/v.
 5. The composition of claim 1,wherein an effective amount of benzethonium chloride is a concentrationof from about 0.01 to about 0.1% w/v.
 6. The composition of claim 1,wherein an effective amount of benzethonium chloride is a concentrationof 0.005% w/v.
 7. The composition of claim 1, wherein an effectiveamount of benzethonium chloride is a concentration of 0.01% w/v.
 8. Thecomposition of claim 1, wherein an effective amount of benzethoniumchloride is a concentration of 0.02% w/v.
 9. The composition of claim 2,further defined as comprising benzethonium chloride in a concentrationof from about 0.001 to about 1.0% w/v, and phenoxyethanol in aconcentration of from about 0.01 to about 1.0% w/v.
 10. The compositionof claim 2, further defined as comprising benzethonium chloride in aconcentration of from about 0.01 to about 0.1% w/v, and phenoxyethanolin a concentration of from about 0.1 to about 0.75% w/v.
 11. Thecomposition of claim 2, further defined as comprising benzethoniumchloride in a concentration of 0.005% w/v, and phenoxyethanol in aconcentration of 0.25% w/v.
 12. The composition of claim 2, furtherdefined as comprising benzethonium chloride in a concentration of 0.005%w/v, and phenoxyethanol in a concentration of 0.5% w/v.
 13. Thecomposition of claim 2, further defined as comprising benzethoniumchloride in a concentration of about 0.01% w/v and phenoxyethanol in aconcentration of about 0.5% w/v.
 14. The composition of claim 3, furtherdefined as comprising benzethonium chloride in a concentration of 0.02%w/v, and phenylethyl alcohol in a concentration of 0.25% w/v.
 15. Thecomposition of claim 3, further defined as comprising about 0.02% w/vbenzethonium chloride and about 0.25% w/v phenylethyl alcohol.
 16. Thecomposition of claim 1, further defined as comprising a salt.
 17. Thecomposition of claim 16, wherein said salt is sodium chloride.
 18. Thecomposition of claim 1, further defined as comprising a buffer.
 19. Thecomposition of claim 18, wherein said buffer is sodium phosphate.
 20. Anerythropoietin pharmaceutical carrier composition, wherein said carrierprovides a stable, sterile, multi-dose formulation for erythropoietinand comprises an amount of benzethonium chloride effective to inhibitmicrobial growth in said composition.
 21. The pharmaceutical carrier ofclaim 20, further comprising phenoxyethanol.
 22. The pharmaceuticalcarrier of claim 20, further comprising phenylethyl alcohol.
 23. Thepharmaceutical carrier of claim 20, wherein an effective amount ofbenzethonium chloride is a concentration of from about 0.001 to about1.0% w/v.
 24. The pharmaceutical carrier of claim 20, wherein aneffective amount of benzethonium chloride is a concentration of fromabout 0.01 to about 0.1% w/v.
 25. The pharmaceutical carrier of claim20, wherein an effective amount of benzethonium chloride is aconcentration of 0.005% w/v.
 26. The pharmaceutical carrier of claim 20,wherein an effective amount of benzethonium chloride is a concentrationof 0.01% w/v.
 27. The pharmaceutical carrier of claim 20, wherein aneffective amount of benzethonium chloride is a concentration of 0.02%w/v.
 28. The pharmaceutical carrier of claim 21, further defined ascomprising benzethonium chloride in a concentration of from about 0.001to about 1.0% w/v, and phenoxyethanol in a concentration of from about0.01 to about 1.0% w/v.
 29. The pharmaceutical carrier of claim 21,further defined as comprising benzethonium chloride in a concentrationof from about 0.01 to about 0.1% w/v, and phenoxyethanol in aconcentration of from about 0.1 to about 0.75% w/v.
 30. Thepharmaceutical carrier of claim 21, further defined as comprisingbenzethonium chloride in a concentration of 0.005% w/v, andphenoxyethanol in a concentration of 0.25% w/v.
 31. The pharmaceuticalcarrier of claim 21, further defined as comprising benzethonium chloridein a concentration of 0.005% w/v, and phenoxyethanol in a concentrationof 0.5% w/v.
 32. The pharmaceutical carrier of claim 21, further definedas comprising benzethonium chloride in a concentration of about 0.01%w/v, and phenoxyethanol in a concentration of about 0.5% w/v.
 33. Thepharmaceutical carrier of claim 22, further defined as comprisingbenzethonium chloride in a concentration of 0.02% w/v, and phenylethylalcohol in a concentration of 0.25% w/v.
 34. The pharmaceutical carrierof claim 20, further comprising one or more additives selected from thegroup consisting of a buffer, a salt, and anti-adsorbent, and asurfactant.
 35. A vial for containing multiple dosages oferythropoietin, wherein said vial contains a solution comprisingerythropoietin and an amount of benzethonium chloride effective toinhibit microbial growth in said composition.
 36. The vial of claim 35,wherein said solution further comprises phenoxyethanol.
 37. The vial ofclaim 35, wherein said solution further comprises phenylethyl alcohol.38. The vial of claim 35, wherein an effective amount of benzethoniumchloride is a concentration of from about 0.001 to about 1.0% w/v. 39.The vial of claim 35, wherein an effective amount of benzethoniumchloride is a concentration of from about 0.01 to about 0.1% w/v. 40.The vial of claim 35, wherein an effective amount of benzethoniumchloride is a concentration of 0.005% w/v.
 41. The vial of claim 35,wherein an effective amount of benzethonium chloride is a concentrationof 0.01% w/v.
 42. The vial of claim 35, wherein an effective amount ofbenzethonium chloride is a concentration of 0.02% w/v.
 43. The vial ofclaim 36, further defined as comprising benzethonium chloride in aconcentration of from about 0.001 to about 1.0% w/v, and phenoxyethanolin a concentration of from about 0.01 to about 1.0% w/v.
 44. The vial ofclaim 36, further defined as comprising benzethonium chloride in aconcentration of from about 0.01 to about 0.1% w/v, and phenoxyethanolin a concentration of from about 0.1 to about 0.75% w/v.
 45. The vial ofclaim 36, further defined as comprising benzethonium chloride in aconcentration of 0.005% w/v, and phenoxyethanol in a concentration of0.25% w/v.
 46. The vial of claim 36, further defined as comprisingbenzethonium chloride in a concentration of 0.005% w/v, andphenoxyethanol in a concentration of 0.5% w/v.
 47. The vial of claim 36,further defined as comprising benzethonium chloride in a concentrationof about 0.01% w/v, and phenoxyethanol in a concentration of about 0.5%w/v.
 48. The vial of claim 37, further defined as comprisingbenzethonium chloride in a concentration of 0.02% w/v, and phenylethylalcohol in a concentration of 0.25% w/v.
 49. The vial of claim 37,further defined as comprising about 0.02% w/v benzethonium chloride andabout 0.25% w/v phenylethyl alcohol.
 50. The vial of claim 35, whereinsaid solution further comprises a salt.
 51. The vial of claim 50,wherein said salt is sodium chloride.
 52. The vial of claim 35, whereinsaid solution further comprises a buffer.
 53. The vial of claim 52,wherein said buffer is sodium phosphate.
 54. A method of inhibitingmicrobial growth in a solution comprising erythropoietin, said methodcomprising adding benzethonium chloride to said solution.
 55. The methodof claim 54, wherein said method further comprises adding phenoxyethanolto said solution.
 56. The method of claim 54, wherein said methodfurther comprises adding phenylethyl alcohol to said solution.
 57. Themethod of claim 54, wherein said benzethonium chloride is added to aconcentration of from about 0.001 to about 1.0% w/v.
 58. The method ofclaim 54, wherein said benzethonium chloride is added to a concentrationof from about 0.01 to about 0.1% w/v.
 59. The method of claim 54,wherein said benzethonium chloride is added to a concentration of 0.005%w/v.
 60. The method of claim 54, wherein said benzethonium chloride isadded to a concentration of 0.01% w/v.
 61. The method of claim 54,wherein said benzethonium chloride is added to a concentration of 0.02%w/v.
 62. The method of claim 55, wherein benzethonium chloride is addedin a concentration of from about 0.001 to about 1.0% w/v, andphenoxyethanol is added in a concentration of from about 0.01 to about1.0% w/v.
 63. The method of claim 55, wherein benzethonium chloride isadded in a concentration of from about 0.01 to about 0.1% w/v, andphenoxyethanol is added in a concentration of from about 0.1 to about0.75% w/v.
 64. The method of claim 55, wherein benzethonium chloride isadded in a concentration of 0.005% w/v, and phenoxyethanol is added in aconcentration of 0.25% w/v.
 65. The method of claim 55, whereinbenzethonium chloride is added in a concentration of 0.005% w/v, andphenoxyethanol is added in a concentration of 0.5% w/v.
 66. The methodof claim 55, wherein benzethonium chloride is added in a concentrationof about 0.01% w/v, and phenoxyethanol is added in a concentration ofabout 0.5% w/v.
 67. The method of claim 56, wherein benzethoniumchloride is added in a concentration of 0.02% w/v, and phenylethylalcohol is added in a concentration of 0.25% w/v.
 68. The method ofclaim 56, wherein benzethonium chloride is added in a concentration ofabout 0.02% w/v, and henylethyl alcohol is added in a concentration ofabout 0.25% w/v.
 69. The method of claim 54, further comprising adding asalt to said solution.
 70. The method of claim 69, wherein said salt issodium chloride.
 71. The method of claim 54, further comprising adding abuffer to said solution.
 72. The method of claim 71, wherein said bufferis sodium phosphate.